Axmi115 variant insecticidal gene and methods for its use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. The toxin coding sequences can be used in DNA constructs or expression cassettes for expression in plants and bacteria. Compositions include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, polynucleotide sequences and the toxin proteins encoded thereby are provided. Also provided are antibodies specifically binding to those amino acid sequences. In particular, the invention encompasses nucleotide sequences encoding fusion proteins, as well as biologically active variants and fragments thereof, wherein the fusion protein contains the C-terminal portion of SEQ ID NO:43. The fusion protein may also contain the N-terminal portion of SEQ ID NO:45. The invention also includes the nucleotide sequence of SEQ ID NO:47 and 1-14, or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:2, 3, 7-14 and 47, including biologically active variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 13/439,269,filed Apr. 4, 2012, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/471,848, filed Apr. 5, 2011, the contents ofwhich are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA116021USSEQLIST.txt”, created on Sep. 11, 2015, and having a size of241 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Hemipteran, Dipteran, and Coleopteran larvae. These proteins also haveshown activity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga,and Acari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) The crystal proteindoes not exhibit insecticidal activity until it has been ingested andsolubilized in the insect midgut. The ingested protoxin is hydrolyzed byproteases in the insect digestive tract to an active toxic molecule.(Höfte and Whiteley (1989) Microbiol. Rev. 53:242-255). This toxin bindsto apical brush border receptors in the midgut of the target larvae andinserts into the apical membrane creating ion channels or pores,resulting in larval death.

In addition to the endotoxins, B. thuringiensis also produces secretedinsecticidal proteins during its vegetative growth stage, namely,vegetative insecticidal proteins (Vip). Since the discovery of the firstVip toxin, two major groups of Vip toxins have been identified in B.thuringiensis. One group of Vip toxins consists of binary toxins whichare made of two components, Vip1 and Vip2 (Warren (1997) In N. B.Carozzi and M. G. Koziel (ed.), Advances in insect control: the role oftransgenic plants. Taylor & Francis, London, United Kingdom). Thecombination of Vip1 and Vip2 is highly insecticidal to an agriculturallyimportant insect, the western corn rootworm (Diabrotica virgifera), butdoes not show any insecticidal activity for any lepidopteran insects(Han et al. (1999) Nat. Struct. Biol. 6:932-936). The other groupconsists of Vip3 toxins, which share no sequence similarity to Vip1 orVip2. The first-identified Vip3 toxin, Vip3Aa1, is highly insecticidalto several major lepidopteran pests of maize and cotton, including thefall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpazea, but shows no activity against the European corn borer Ostrinianubilalis, a major pest of maize (Estruch et al. (1996) Proc. Natl.Acad. Sci. USA 93:5389-5394). The deletion of the vip3Aa1 gene from a B.thuringiensis strain resulted in a significant reduction of theinsecticidal activity of that B. thuringiensis strain, suggesting thatVip3 contributes to the overall toxicity of B. thuringiensis strains(Donovan et al. (2001) J. Invertebr. Pathol. 78:45-51). It was alsoobserved that Vip3Aa1 kills insects by lysing insect midgut cells (Yu etal. (1997) Appl. Environ. Microbiol. 63:532-536) via cell membrane poreformation (Lee et al. (2003) Appl. Environ. Microbiol. 69:4648-4657).

The intensive use of B. thuringiensis-based insecticides has alreadygiven rise to resistance in field populations of the diamondback moth,Plutella xylostella (Ferre and Van Rie (2002) Annu. Rev. Entomol.47:501-533). The most common mechanism of resistance is the reduction ofbinding of the toxin to its specific midgut receptor(s). This may alsoconfer cross-resistance to other toxins that share the same receptor(Ferre and Van Rie (2002)).

SUMMARY OF INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsectidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleotide sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise bacteria, plants, plant cells,tissues, and seeds comprising the nucleotide sequence of the invention.

In particular, isolated nucleic acid molecules are provided that encodea pesticidal protein. Additionally, amino acid sequences correspondingto the pesticidal protein are encompassed. In particular, the presentinvention provides for an isolated or recombinant nucleic acid moleculecomprising a nucleotide sequence encoding a fusion protein, as well asbiologically active variants and fragments thereof, wherein the fusionprotein comprises the C-terminal portion of SEQ ID NO:43. In variousembodiments, the fusion protein comprises the N-terminal portion of SEQID NO:45. In specific embodiments, the nucleic acid molecule encompassedby the present invention (including vectors, host cells, plants, andseeds comprising the nucleic acid molecule) comprises the nucleotidesequence set forth in SEQ ID NO:47 and 1-14, or a nucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NO:2, 3, 7-14 and47, including biologically active variants and fragments thereof.Nucleotide sequences that are complementary to a nucleotide sequence ofthe invention, or that hybridize to a sequence of the invention or acomplement thereof are also encompassed. Isolated or recombinant fusionproteins encoded by the nucleci acid molecule of the invention are alsoencompassed herein.

Methods are provided for producing the polypeptides of the invention,and for using those polypeptides for controlling or killing alepidopteran, hemipteran, coleopteran, nematode, or dipteran pest.Methods and kits for detecting the nucleic acids and polypeptides of theinvention in a sample are also included.

The compositions and methods of the invention are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the invention are alsouseful for generating altered or improved proteins that have pesticidalactivity, or for detecting the presence of pesticidal proteins ornucleic acids in products or organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram of the fusion constructs.

FIG. 2 shows the results of the in vitro leaf disk bioassay. pAG6585contains optAxmi115v01 (N=14) and pAG6141 contains optAxmi115v02.01.01(N=8).

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance or tolerance in organisms, particularlyplants or plant cells. By “resistance” is intended that the pest (e.g.,insect) is killed upon ingestion or other contact with the polypeptidesof the invention. By “tolerance” is intended an impairment or reductionin the movement, feeding, reproduction, or other functions of the pest.The methods involve transforming organisms with a nucleotide sequenceencoding a pesticidal protein of the invention. In particular, thenucleotide sequences of the invention are useful for preparing plantsand microorganisms that possess pesticidal activity. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.Compositions are pesticidal nucleic acids and proteins of Bacillus orother species. The sequences find use in the construction of expressionvectors for subsequent transformation into organisms of interest, asprobes for the isolation of other homologous (or partially homologous)genes, and for the generation of altered pesticidal proteins by methodsknown in the art, such as domain swapping or DNA shuffling, for example,with members of the Vip1, Vip2, or Vip3 families of toxins. The proteinsfind use in controlling or killing lepidopteran, hemipteran,coleopteran, dipteran, and nematode pest populations and for producingcompositions with pesticidal activity.

By “pesticidal toxin” or “pesticidal protein” is intended a toxin thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, and Coleoptera orders, or theNematoda phylum, or a protein that has homology to such a protein.Pesticidal proteins have been isolated from organisms including, forexample, Bacillus sp., Clostridium bifermentans and Paenibacilluspopilliae. Pesticidal proteins include amino acid sequences deduced fromthe full-length nucleotide sequences disclosed herein, and amino acidsequences that are shorter than the full-length sequences, either due tothe use of an alternate downstream start site, or due to processing thatproduces a shorter protein having pesticidal activity. Processing mayoccur in the organism the protein is expressed in, or in the pest afteringestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleotidesequences that confer pesticidal activity. These nucleotide sequencesencode polypeptides with homology to known toxins. Also provided are theamino acid sequences of the pesticidal proteins. The protein resultingfrom translation of this gene allows cells to control or kill pests thatingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding pesticidalproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. Also encompassed herein are nucleotide sequencescapable of hybridizing to the nucleotide sequences of the inventionunder stringent conditions as defined elsewhere herein. As used herein,the term “nucleic acid molecule” is intended to include DNA molecules(e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g.,mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” or “recombinant” nucleic acid sequence (or DNA) is usedherein to refer to a nucleic acid sequence (or DNA) that is no longer inits natural environment, for example in an in vitro or in a recombinantbacterial or plant host cell. In some embodiments, an isolated orrecombinant nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the invention, “isolated” when used to refer to nucleic acidmolecules excludes isolated chromosomes. For example, in variousembodiments, the isolated delta-endotoxin encoding nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1kb of nucleotide sequences that naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. In various embodiments, a delta-endotoxin protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-delta-endotoxin protein (also referred to herein as a “contaminatingprotein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:47 and 1-14, and variants,fragments, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequences for the pesticidal proteins encoded by these nucleotidesequences are set forth in SEQ ID NO:2, 3, 7-14 and 47.

Nucleic acid molecules that are fragments of these nucleotide sequencesencoding pesticidal proteins are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a pesticidal protein. A fragment of a nucleotidesequence may encode a biologically active portion of a pesticidalprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a nucleotide sequence encoding apesticidal protein comprise at least about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthnucleotide sequence encoding a pesticidal protein disclosed herein,depending upon the intended use. By “contiguous” nucleotides is intendednucleotide residues that are immediately adjacent to one another.Fragments of the nucleotide sequences of the present invention willencode protein fragments that retain the biological activity of thepesticidal protein and, hence, retain pesticidal activity. Thus,biologically-active fragments of the polypeptides disclosed herein arealso encompassed. By “retains activity” is intended that the fragmentwill have at least about 30%, at least about 50%, at least about 70%,80%, 90%, 95% or higher of the pesticidal activity of the pesticidalprotein. In various embodiments, the activity may be improved orextended relative to a reference pesticidal protein (e.g., improved orextended relative to the activity of SEQ ID NO:43 or 45) as definedelsewhere herein. In one embodiment, the pesticidal activity iscoleoptericidal activity. In another embodiment, the pesticidal activityis lepidoptericidal activity. In another embodiment, the pesticidalactivity is nematocidal activity. In another embodiment, the pesticidalactivity is diptericidal activity. In another embodiment, the pesticidalactivity is hemiptericidal activity. Methods for measuring pesticidalactivity are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

A fragment of a nucleotide sequence encoding a pesticidal protein thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450 contiguous amino acids, or up to the total number ofamino acids present in a full-length pesticidal protein of theinvention. In some embodiments, the fragment is a proteolytic cleavagefragment. For example, the proteolytic cleavage fragment may have anN-terminal or a C-terminal truncation of at least about 100 amino acids,about 120, about 130, about 140, about 150, or about 160 amino acidsrelative to SEQ ID NO:2, 3, 7-14 and 47. In some embodiments, thefragments encompassed herein result from the removal of the C-terminalcrystallization domain, e.g., by proteolysis or by insertion of a stopcodon in the coding sequence. In other embodiments, the fusion proteincomprises a fragment of the C-terminal domain of SEQ ID NO:43 and/or afragment of the N-terminal domain of SEQ ID NO:45.

Preferred pesticidal proteins of the present invention are encoded by anucleotide sequence sufficiently identical to the nucleotide sequence ofSEQ ID NO:47 and 1-14, or the pesticidal proteins are sufficientlyidentical to the amino acid sequence set forth in SEQ ID NO:2, 3, 7-14and 47. In another embodiment, the nucleotide sequence encodes a fusionprotein, wherein the N-terminal portion is sufficiently identical to theN-terminal portion of SEQ ID NO:45, or wherein the N-terminal portion issufficiently identical to the N-terminal portion of SEQ ID NO:45 and theC-terminal portion is sufficiently identical to SEQ ID NO:43. By“sufficiently identical” is intended an amino acid or nucleotidesequence that has at least about 60% or 65% sequence identity, about 70%or 75% sequence identity, about 80% or 85% sequence identity, about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the percent identity iscalculated across the entirety of the reference sequence (i.e., thesequence disclosed herein as any of SEQ ID NO:1-31, 47 or 48). Thepercent identity between two sequences can be determined usingtechniques similar to those described below, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other, is regarded as a positionwith non-identical residues.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous topesticidal-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the pesticidal protein encoding nucleotide sequencesinclude those sequences that encode the pesticidal proteins disclosedherein but that differ conservatively because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the pesticidal proteinsdisclosed in the present invention as discussed below. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, pesticidal activity. By “retains activity” is intendedthat the variant will have at least about 30%, at least about 50%, atleast about 70%, or at least about 80% of the pesticidal activity of thenative protein. In some embodiments, the activity is improved orextended relative to a reference protein as defined elsewhere herein.Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedpesticidal proteins, without altering the biological activity of theproteins. Thus, variant isolated nucleic acid molecules can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a pesticidal protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the invention (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theinvention (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingpesticidal sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the pesticidal nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known pesticidalprotein-encoding nucleotide sequence disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, at least about 25, at least about 50, 75, 100, 125, 150,175, or 200 consecutive nucleotides of nucleotide sequence encoding apesticidal protein of the invention or a fragment or variant thereof.Methods for the preparation of probes for hybridization are generallyknown in the art and are disclosed in Sambrook and Russell, 2001, supraherein incorporated by reference.

For example, an entire pesticidal sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding pesticidal protein-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Thus, the present invention encompasses probes for hybridization, aswell as nucleotide sequences capable of hybridization to all or aportion of a nucleotide sequence of the invention (e.g., at least about300 nucleotides, at least about 400, at least about 500, 1000, 1200,1500, 2000, 2500, 3000, 3500, or up to the full length of a nucleotidesequence disclosed herein). Hybridization of such sequences may becarried out under stringent conditions. By “stringent conditions” or“stringent hybridization conditions” is intended conditions under whicha probe will hybridize to its target sequence to a detectably greaterdegree than to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Pesticidal proteins are also encompassed within the present invention.By “pesticidal protein” is intended a protein having the amino acidsequence set forth in SEQ ID NO:2, 3, 7-14 and 47. Fragments,biologically active portions, and variants thereof are also provided,and may be used to practice the methods of the present invention. An“isolated protein” or a “recombinant protein” is used to refer to aprotein that is no longer in its natural environment, for example invitro or in a recombinant bacterial or plant host cell.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO:2, 3, 7-14 and 47, and thatexhibit pesticidal activity. A biologically active portion of apesticidal protein can be a polypeptide that is, for example, 10, 25,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,or more amino acids in length. Such biologically active portions can beprepared by recombinant techniques and evaluated for pesticidalactivity. Methods for measuring pesticidal activity are well known inthe art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. As used here, a fragment comprises at least 8 contiguous aminoacids of SEQ ID NO:2, 3, 7-14 and 47. The invention encompasses otherfragments, however, such as any fragment in the protein greater thanabout 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350 or more amino acids in length.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of any of SEQ ID NO:2, 3, 7-14 and 47.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NO:47 and 1-14,or a complement thereof, under stringent conditions. Variants includepolypeptides that differ in amino acid sequence due to mutagenesis.Variant proteins encompassed by the present invention are biologicallyactive, that is they continue to possess the desired biological activityof the native protein, that is, retaining pesticidal activity. In someembodiments, the variants have improved activity relative to the nativeprotein. Methods for measuring pesticidal activity are well known in theart. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. On rare occasions, translation in bacterial systemscan initiate at a TTG codon, though in this event the TTG encodes amethionine. Furthermore, it is not often determined a priori which ofthese codons are used naturally in the bacterium. Thus, it is understoodthat use of one of the alternate methionine codons may also lead togeneration of pesticidal proteins. These pesticidal proteins areencompassed in the present invention and may be used in the methods ofthe present invention. It will be understood that, when expressed inplants, it will be necessary to alter the alternate start codon to ATGfor proper translation.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a pesticidal protein may bealtered by various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a pesticidal protein of the present invention. Thisprotein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions of one or moreamino acids of SEQ ID NO:2, 3, 7-14 and 47, including up to about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 15, about 20, about 25, about 30, about 35, about 40, about 45,about 50, about 55, about 60, about 65, about 70, about 75, about 80,about 85, about 90, about 100, about 105, about 110, about 115, about120, about 125, about 130, about 135, about 140, about 145, about 150,about 155, or more amino acid substitutions, deletions or insertionswithin either the C-terminal portion or the N-terminal portion, or both.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a pesticidal protein can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of a pesticidal protein to confer pesticidal activity may beimproved by the use of such techniques upon the compositions of thisinvention. For example, one may express a pesticidal protein in hostcells that exhibit high rates of base misincorporation during DNAreplication, such as XL-1 Red (Stratagene, La Jolla, Calif.). Afterpropagation in such strains, one can isolate the DNA (for example bypreparing plasmid DNA, or by amplifying by PCR and cloning the resultingPCR fragment into a vector), culture the pesticidal protein mutations ina non-mutagenic strain, and identify mutated genes with pesticidalactivity, for example by performing an assay to test for pesticidalactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests. Examples of mutations that result in increasedtoxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev.62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent pesticidal protein coding regions can be used to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneof the invention and other known pesticidal genes to obtain a new genecoding for a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpesticidal proteins. Domains may be swapped between pesticidal proteins,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; deMaagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al.(1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol.Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol.65:2918-2925).

Thus, in various embodiments of the present invention, the nucleic acidsequences encompassed herein (as well as compositions, vectors, hostcells, plants, and seed comprising the nucleic acid sequence) comprise aportion of one or more toxin(s) and a portion of one of more differenttoxin(s). In one embodiment, the nucleic acid sequence comprises anucleotide sequence encoding the N-terminal portion of Axmi005 (which isset forth in SEQ ID NO:45) and the C-terminal portion of Axmi115 (whichis set forth in SEQ ID NO:43). In specific embodiments, the N-terminalportion of Axmi005 comprises from about amino acid residues 1 to 173, orfrom about amino acid residue 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, or 50 to about amino acid residue 150, 155, 160, 165,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 250, 300, 325, or 350 of Axmi005 andthe C-terminal portion of Axmi115 comprises from about amino acidresidue 174 to about amino acid residue 803 of Axmi115, or from aboutamino acid residue 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 250, 300, 325, or350 to about amino acid residue 600, 650, 700, 750, 760, 770, 780, 790,795, 796, 797, 798, 799, 800, 801, 802, or 803. One of skill in the artwill recognize that minor variants and deletions within each of theamino acid sequences can be made and still retain (or improve) activityof the fusion protein. In some embodiments, the nucleic acid sequencesof the invention encode an Axmi005/Axmi115 fusion protein with amutation (relative to the corresponding region of the parent Axmi005 orAxmi115 protein) at one or more of positions corresponding to the aminoacid residues at positions 584, 588, and 771 relative to SEQ ID NO:43(see, for example, the variant fusion sequences found in SEQ IDNO:18-22). In other embodiments, the nucleotide sequence encompassedherein is set forth in any of SEQ ID NO:47 and 1-14 and the amino acidsequence is set forth in any of SEQ ID NO:2, 3, 7-14 and 47.

In various embodiments, the fusion of Axmi005 with Axmi115 results in anamino acid sequence having improved or extended activity compared to theactivity of either Axmi005 or Axmi115 alone. By “improved” activity isintended an increase in death to at least one pest, or an increase inthe noticeable reduction of pest growth, feeding, or normalphysiological development relative to the native protein. By “extended”activity is intended activity against a pest that was not demonstratedby both Axmi005 and Axmi115. For example, fusion of a portion of Axmi005with a portion of Axmi115 could result in a single protein having theactivity profile of both Axmi005 and Axmi115. In some embodiments,activity against an individual pest is improved in the fusion proteinover one or both of Axmi005 and/or Axmi115.

Vectors

A pesticidal sequence of the invention may be provided in an expressioncassette for expression in a plant of interest. By “plant expressioncassette” is intended a DNA construct that is capable of resulting inthe expression of a protein from an open reading frame in a plant cell.Typically these contain a promoter and a coding sequence. Often, suchconstructs will also contain a 3′ untranslated region. Such constructsmay contain a “signal sequence” or “leader sequence” to facilitateco-translational or post-translational transport of the peptide tocertain intracellular structures such as the chloroplast (or otherplastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.Insecticidal toxins of bacteria are often synthesized as protoxins,which are protolytically activated in the gut of the target pest (Chang(1987) Methods Enzymol. 153:507-516). In some embodiments of the presentinvention, the signal sequence is located in the native sequence, or maybe derived from a sequence of the invention. By “leader sequence” isintended any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and/or 3′ regulatory sequences operably linkedto a sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

In various embodiments, the nucleotide sequence of the invention isoperably linked to a promoter, e.g., a plant promoter. “Promoter” refersto a nucleic acid sequence that functions to direct transcription of adownstream coding sequence. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary for the expression of aDNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the pesticidal sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, U.S. Patent Publication No. 20090137409,and Murray et al. (1989) Nucleic Acids Res. 17:477-498, hereinincorporated by reference.

In one embodiment, the pesticidal protein is targeted to the chloroplastfor expression. In this manner, where the pesticidal protein is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe pesticidal protein to the chloroplasts. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481.

The pesticidal gene to be targeted to the chloroplast may be optimizedfor expression in the chloroplast to account for differences in codonusage between the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

The transgenic plants of the invention express one or more of the noveltoxin sequences disclosed herein. In various embodiments, the transgenicplant further comprises one or more additional genes for insectresistance (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C,Cry1D, Cry1E, and Cry1F families; Cry2, such as members of the Cry2Afamily; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E,and Cry9F families; etc.). It will be understood by one of skill in theart that the transgenic plant may comprise any gene imparting anagronomic trait of interest.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The pesticidal gene of the invention may bemodified to obtain or enhance expression in plant cells. Typically aconstruct that expresses such a protein would contain a promoter todrive transcription of the gene, as well as a 3′ untranslated region toallow transcription termination and polyadenylation. The organization ofsuch constructs is well known in the art. In some instances, it may beuseful to engineer the gene such that the resulting peptide is secreted,or otherwise targeted within the plant cell. For example, the gene canbe engineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. It may also be preferable toengineer the plant expression cassette to contain an intron, such thatmRNA processing of the intron is required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors.” Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the pesticidal gene are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thepesticidal protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a pesticidal protein that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a pesticidal protein may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a pesticidal protein may be tested forpesticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay for pestactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape., etc.).

Use in Pesticidal Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pest control or inengineering other organisms as pesticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene of the invention andprotein may be used for protecting agricultural crops and products frompests. In one aspect of the invention, whole, i.e., unlysed, cells of atoxin (pesticide)-producing organism are treated with reagents thatprolong the activity of the toxin produced in the cell when the cell isapplied to the environment of target pest(s).

Alternatively, the pesticide is produced by introducing a pesticidalgene into a cellular host. Expression of the pesticidal gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. In one aspect of this invention, these cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of the targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated pesticides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein. Alternatively,one may formulate the cells expressing a gene of this invention such asto allow application of the resulting material as a pesticide.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, hemipteran, dipteran, or coleopteran pests may be killedor reduced in numbers in a given area by the methods of the invention,or may be prophylactically applied to an environmental area to preventinfestation by a susceptible pest. Preferably the pest ingests, or iscontacted with, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, the crystal and/or the spore suspension, or theisolated protein component with the desired agriculturally-acceptablecarrier. The compositions may be formulated prior to administration inan appropriate means such as lyophilized, freeze-dried, desiccated, orin an aqueous carrier, medium or suitable diluent, such as saline orother buffer. The formulated compositions may be in the form of a dustor granular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in pesticide formulationtechnology; these are well known to those skilled in pesticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the pesticidal composition withsuitable adjuvants using conventional formulation techniques. Suitableformulations and application methods are described in U.S. Pat. No.6,468,523, herein incorporated by reference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrinidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylinidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with (or susceptible toinfestation by) a pest against which said polypeptide has pesticidalactivity. In some embodiments, the polypeptide has pesticidal activityagainst a lepidopteran, coleopteran, dipteran, hemipteran, or nematodepest, and said field is infested with a lepidopteran, hemipteran,coleopteran, dipteran, or nematode pest. As defined herein, the “yield”of the plant refers to the quality and/or quantity of biomass producedby the plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence. In specific methods, plant yield is increased as aresult of improved pest resistance of a plant expressing a pesticidalprotein disclosed herein. Expression of the pesticidal protein resultsin a reduced ability of a pest to infest or feed.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides. Exemplarychemical compositions include: Fruits/Vegetables Herbicides: Atrazine,Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate,Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron,Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb,Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin,Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb,Emamectin-benzoate, Indoxacarb, Fenamiphos, Pyriproxifen,Fenbutatin-oxid; Fruits/Vegetables Fungicides: Ametoctradin,Azoxystrobin, Benthiavalicarb, Boscalid, Captan, Carbendazim,Chlorothalonil, Copper, Cyazofamid, Cyflufenamid, Cymoxanil,Cyproconazole, Cyprodinil, Difenoconazole, Dimetomorph, Dithianon,Fenamidone, Fenhexamid, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram,Fluoxastrobin, Fluxapyroxad, Folpet, Fosetyl, Iprodione, Iprovalicarb,Isopyrazam, Kresoxim-methyl, Mancozeb, Mandipropamid,Metalaxyl/mefenoxam, Metiram, Metrafenone, Myclobutanil, Penconazole,Penthiopyrad, Picoxystrobin, Propamocarb, Propiconazole, Propineb,Proquinazid, Prothioconazole, Pyraclostrobin, Pyrimethanil, Quinoxyfen,Spiroxamine, Sulphur, Tebuconazole, Thiophanate-methyl, Trifloxystrobin;Cereals Herbicides: 2.4-D, Amidosulfuron, Bromoxynil, Carfentrazone-E,Chlorotoluron, Chlorsulfuron, Clodinafop-P, Clopyralid, Dicamba,Diclofop-M, Diflufenican, Fenoxaprop, Florasulam, Flucarbazone-NA,Flufenacet, Flupyrosulfuron-M, Fluroxypyr, Flurtamone, Glyphosate,Iodosulfuron, Ioxynil, Isoproturon, MCPA, Mesosulfuron, Metsulfuron,Pendimethalin, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyroxsulam,Sulfosulfuron, Thifensulfuron, Tralkoxydim, Triasulfuron, Tribenuron,Trifluralin, Tritosulfuron; Cereals Fungicides: Azoxystrobin, Bixafen,Boscalid, Carbendazim, Chlorothalonil, Cyflufenamid, Cyproconazole,Cyprodinil, Dimoxystrobin, Epoxiconazole, Fenpropidin, Fenpropimorph,Fluopyram, Fluoxastrobin, Fluquinconazole, Fluxapyroxad, Isopyrazam,Kresoxim-methyl, Metconazole, Metrafenone, Penthiopyrad, Picoxystrobin,Prochloraz, Propiconazole, Proquinazid, Prothioconazole, Pyraclostrobin,Quinoxyfen, Spiroxamine, Tebuconazole, Thiophanate-methyl,Trifloxystrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin,Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin,Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Clorphyriphos, Pirimicarb, Methiocarb, Sulfoxaflor; MaizeHerbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba,Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole,(S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron,Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil,Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides:Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid,Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin,Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin,Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin; Maize Fungicides: Azoxystrobin, Bixafen,Boscalid, Cyproconazole, Dimoxystrobin, Epoxiconazole, Fenitropan,Fluopyram, Fluoxastrobin, Fluxapyroxad, Isopyrazam, Metconazole,Penthiopyrad, Picoxystrobin, Propiconazole, Prothioconazole,Pyraclostrobin, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenobucarb, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr,Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Etofenprox, Carbofuran,Benfuracarb, Sulfoxaflor; Rice Fungicides: Azoxystrobin, Carbendazim,Carpropamid, Diclocymet, Difenoconazole, Edifenphos, Ferimzone,Gentamycin, Hexaconazole, Hymexazol, Iprobenfos (IBP), Isoprothiolane,Isotianil, Kasugamycin, Mancozeb, Metominostrobin, Orysastrobin,Pencycuron, Probenazole, Propiconazole, Propineb, Pyroquilon,Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole,Trifloxystrobin, Validamycin; Cotton Herbicides: Diuron, Fluometuron,MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim,Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin,Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate,Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb,Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor; Cotton Fungicides: Azoxystrobin, Bixafen, Boscalid,Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole,Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram,Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb,Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb,Prothioconazole, Pyraclostrobin, Quintozene, Tebuconazole,Tetraconazole, Thiophanate-methyl, Trifloxystrobin; Soybean Herbicides:Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-) Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin,gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Bixafen, Boscalid,Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole,Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin,Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil, Mancozeb,Maneb, Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad,Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin,Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin;Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate,Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron,Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop;Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin,gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Bixafen,Boscalid, Carbendazim, Cyproconazole, Difenoconazole, Dimoxystrobin,Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flusilazole,Fluxapyroxad, Iprodione, Isopyrazam, Mepiquat-chloride, Metconazole,Metominostrobin, Paclobutrazole, Penthiopyrad., Picoxystrobin,Prochloraz, Prothioconazole, Pyraclostrobin, Tebuconazole,Thiophanate-methyl, Trifloxystrobin, Vinclozolin; Canola Insecticides:Carbofuran, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin,Thiamethoxam, Acetamiprid, Dinetofuran, β-Cyfluthrin, gamma and lambdaCyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram,Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLES Example 1 Design and Testing of Axmi115 FusionProteins

Axmi115 is described in U.S. Patent Publication 20100004176 (the aminoacid sequence is set forth herein as SEQ ID NO:43). This gene shares 70%sequence homology with Vip3Aa. A codon optimized version of Axmi115(also referred to herein as Axmi115v01 and set forth in SEQ ID NO:42)was cloned and expressed using the E. coli expression vector. Theprotein produced was shown in in vitro bioassay to have pesticidalactivity against various insect pests including European corn borer(ECB), corn earworm (CEW), fall armyworm (FAW) and black cutworm (BCW).

Axmi005 is also described in U.S. Patent Publication 20100004176. Thisgene shares 94% sequence homology with Vip3Aa. A codon optimized versionof Axmi005 (optAxmi005, which is set forth herein as SEQ ID NO:44) wascloned and expressed using the E. coli expression vector. The proteinproduced was shown in in vitro bioassay to have pesticidal activityagainst various insect pests including Helicoverpa zea (Hz), Heliothisvirescens (Hv), FAW, BCW, sugarcane borer (SCB), and velvetbeancaterpillar (VBC).

The relative activity of Axmi115 was low against Hz and FAW compared toAxmi005. Also as noted above Axmi005 did not have ECB activity. In anattempt to identify the domains responsible for the differentialspecificity as well as activity of the two proteins, constructsexpressing fusions of optAxmi005 and a codon-optimized version ofAxmi115 (optAxmi115v01) were made as described below and diagrammed inFIG. 1. The protein was expressed in E. coli and tested against ECB, Hz,FAW and BCW in in vitro bioassay. The protein expressed by pAX6307(Axmi115v02.01, set forth herein as SEQ ID NO:1) showed enhancedactivity when compared with the protein expressed by pAX5477(Axmi115v01, set forth herein as SEQ ID NO:42) against all four peststested.

The gene expressed in pAX6307 (Axmi115v02.01) was vectored into theplant expression vector pAG6141 in which expression of the protein wasdriven by the Sugar cane Ubiquitin promoter.

Leaf samples from transgenic plants expressing Axmi115v01 andAxmi115v02.01 were tested in laboratory insect bioassay against ECB, Hz,FAW and BCW and in field tests against ECB, Hz and FAW. Results showthat the improved Axmi115v02.01 gene had better efficacy against allpests tested.

Description of Constructs:

Amino acid sequences derived by in silico translation of the DNAsequence of Vip3Aa, Axmi005, Axmi115v01, Axmi163, and Axmi184 werealigned to identify conserved amino acids in all homologs (Axmi163 andAxmi184 are also described in U.S. Patent Publication 20100004176).

PCR primers were designed to three conserved regions of Axmi005 andAxmi115v01 using the sequence of optAxmi005 found in pAX5478 (whichcontains a codon optimized version of Axmi005, set forth in SEQ IDNO:44) and the sequence of optAxmi115 found in pAX5477 (which contains acodon optimized version of Axmi115). Three fusion genes were generatedby overlap PCR (see FIG. 1).

The DNA of the fusion genes produced by these PCR reactions was clonedinto the E. coli expression vector pRSf1B. The resulting expressionvectors are shown in Table 1. Protein was expressed using known methodsand the E. coli extract was tested in an in vitro bioassay.

TABLE 1 Fusion gene constructs Construct Nucleotide Amino acid nameSequence insert SEQ ID NO: SEQ ID NO: pAX6307 Axmi005/Axmi115 1 15fusion A pAX6308 Axmi005/Axmi115 2 16 fusion B pAX6309 Axmi005/Axmi115 317 fusion D pAX5478 optAxmi005 44 45 pAX5477 Axmi115v01 42 43 pRSf1bvector control — —

In-Vitro Bioassay

Crude extracts from E. coli expressed in vectors was assayed against Hz,ECB, FAW, and BCW. The results are shown in Table 2 (stunt) and Table 3(mortality).

TABLE 2 Stunt score ECB Hz FAW BCW ave* SD ave* SD ave* SD ave* SDpAX6307 2.2 0.3 pAX6307 1.8 0.3 pAX6307 3 0 pAX6307 0.8 0.3 (fusion A)pAX6308 0.5 0.5 pAX6308 0.3 0.4 pAX6308 1.3 1.3 pAX6308 0 0 (fusion B)pAX6309 1.2 0.3 pAX6309 0.2 0.3 pAX6309 0.7 0.7 pAX6309 0 0 (fusion D)pRSf1b 0.3 0.4 pRSf1b 0 0 pRSf1b 0.5 0.5 pRSf1b 0 0 (vector control)pAX5478 0 0 pAX5478 1.8 0.3 pAX5478 3 0 pAX5478 1.7 0.7 (Axmi005)pAX5477 0.2 0.3 pAX5477 0.5 0.5 pAX5477 1.5 1.5 pAX5477 0.2 0.3(Axmi115v01) *Scoring system: 0 = no effect observed 1 = mildnon-uniform stunting 2 = moderate non-uniform stunting 3 = moderate tosevere uniform stunting 4 = mortality (<100%) with uniform stunting 5 =complete mortality

TABLE 3 Percent mortality Hz ECB FAW BCW pAX6307 50 50 75 25 (fusion A)pAX6308 0 0 0 0 (fusion B) pAX6309 0 25 25 0 (fusion D) pRSf1b 0 0 0 0(vector control) pAX5478 50 0 75 25 (optAxmi005) pAX5477 0 0 0 0(Axmi115v01)The protein expressed from vector pAX6307 (fusion A) varied by six aminoacids and was designated Axmi115v02.01. The amino acid sequence for thisfusion protein is set forth in SEQ ID NO:15.

E. coli expression vectors expressing Axmi115v01 (pAX5476) andAxmi115v02.01 (pAX6307) had N-terminal 6×His Tags. The two proteins werepurified using the nickel binding properties of the 6×His tag. Variousconcentrations of the purified protein were assayed by in vitro bioassayagainst ECB, FAW, BCW and Beet Armyworm (BAW). The results show thatAxmi115v02.01 has enhanced activity compared with Axmi115v01 in allcases (Tables 4 and 5).

TABLE 4 Stunt score μg/ml BAW FAW ECB BCW Axmi115v01 40 4 4 3 0Axmi115v01 10 1 3 0 0 Axmi115v01 1 0 0 0 0 Axmi115v01 0.1 0 0 0 0Axmi115v01 0.01 0 1 0 0 Axmi115v02 40 4 4 3 3 Axmi115v02 10 4 4 3 1Axmi115v02 1 4 4 3 0 Axmi115v02 0.1 2 1 2 0 Axmi115v02 0.01 0 2 1 0

TABLE 5 Mortality score μg/ml BAW FAW ECB BCW Axmi115v01 40 75%  0% 0%0% Axmi115v01 10 0% 25%  0% 0% Axmi115v01 1 0% 0% 0% 0% Axmi115v01 0.10% 0% 0% 0% Axmi115v01 0.01 0% 0% 0% 0% Axmi115v02 40 75% 50%  0% 0%Axmi115v02 10 0% 25%  50%  0% Axmi115v02 1 0% 25%  0% 0% Axmi115v02 0.10% 0% 0% 0% Axmi115v02 0.01 0% 0% 0% 0%

Plant Leaf Disc Bioassay

Axmi115v01 (SEQ ID NO:42) and Axmi115v02.01 (SEQ ID NO:1) were clonedinto plant expression vectors pAG6585 and pAG6141, respectively, andtransgenic maize plants were produced. Samples were taken for PCR andWestern analysis and for in vitro leaf disc bioassay against Hz, ECB,FAW, and BCW. The bioassay was scored for undamaged, low damage (1-fewholes), moderate damage, and heavy damage. Undamaged and light damagedwere considered a positive result whereas moderate to heavy damage wasconsidered a negative result.

Leaf material from PCR and western positive plants was assayed in invitro leaf disk bioassay. FIG. 2A shows the percent PCR positive plantsthat gave a bioassay score of undamaged, light damage, moderate damageor heavy damage for each construct. Western blots indicate that theexpression level of protein in plants expressing optAxmi115v02.01 is, ingeneral, higher than plants expressing Axmi115v01.

Additional transgenic plants were produced expressing Axmi115v02.01.Leaf material from PCR and Western positive plants was assayed in invitro leaf disk bioassay against Hz, ECB, FAW, and BCW. The results areshown in FIG. 2 b.

Plant Field Trials

Plants expressing the genes shown in Table 6 were planted at the PolkCounty, Iowa test location. Negative segregates were identified andremoved using a 1× application of Glyphosate (20 oz./A of Buccaneer 5,Tenkoz, Inc.) when plants were at the V3-V4 leaf stage. Insect pressureresulted from manual infestations of ECB, Hz, and FAW.

Infestations of ECB mimicked the natural occurrence of first and secondgenerations. For ECB, in total, approximately 340 larvae were infestedinto either the leaf whorls (first generation, ECB1) or leaf axils(second generation, ECB2) of each plant. ECB1 was evaluated by theGuthrie 1-9 rating scale. ECB2 was a measure of the total length ofstalk tunneling measured in cm.

Twenty Hz larvae were infested onto the tips of primary ears on eachplant. There was also a moderate natural infestation of Hz thataugmented these manual infestations. The ear damage was measured in sq.cm.

Approximately 60 FAW larvae were infested into the leaf whorls. Damagewas measured in Modified Davis 1-9 rating scale as described below.

The results of these field trials are shown in Table 6.

TABLE 6 Field trial results FAW (1-9) ECB2 (cm) Mean Hz (sq. cm) MeanScore SD Mean Score SD score SD Axmi115v02.01 1.20 0.48 0.12 0.15 0.000.00 Axmi115v01 1.92 1.18 1.96 1.40 0.83 N/A Axmi005 1.75 0.97 4.12 2.06N/A N/A neg. Control 6.42 0.74 7.06 1.61 9.65 1.66

FAW—Modified Davis 1-9 Rating Scale Description.

1. No visible damage or only pinhole lesions present on whorl leaves.2. Pinhole and small circular lesions present on whorl leaves.3. Small circular lesions and a few small elongated (rectangular-shaped)lesions of up to 1.3 cm (½″) in length present on whorl and furl leaves.4. Several small to mid-sized 1.3 to 2.5 cm (½″ to 1″) in lengthelongated lesions present on a few whorl and furl leaves.5. Several large elongated lesions greater than 2.5 cm (1″) in lengthpresent on a few whorl and furl leaves and/or a few small- to mid-sizeduniform to irregular shaped holes (basement membrane consumed) eatenfrom the whorl and/or furl leaves.6. Several large elongated lesions present on several whorl and furlleaves and/or several large uniform to irregular shaped holes eaten fromfurl/whorl leaves.7. Many elongated lesions of all sizes present on several whorl and furlleaves plus several large uniform to irregular shaped holes eaten fromthe whorl and furl leaves.8. Many elongated lesions of all sizes present on most whorl and furlleaves plus many mid- to large-sized uniform to irregular shaped holeseaten from the whorl and furl leaves.9. Whorl and furl leaves almost totally destroyed.

-   Davis, F. M., S. S. Ng, and W. P. Williams. 1992. Visual rating    scales for screening whorl-stage corn for resistance to fall    armyworm. Miss. Agric. Forestry Exp. Stn. Tech. Bull. 186.

ECB—Guthrie 1-9 Rating Scale Description.

1. No visible leaf injury.

2. Small amount of shot-hole injury on a few leaves.

3. Shot-hole injury common on several leaves.

4. Several leaves with shot-holes and elongated lesions.

5. Several leaves with elongated lesions.

6. Several leaves with elongated lesions about 2.5 cm long.

7. Long lesions common on about one-half of the leaves.

8. Long lesions common on about two-thirds of the leaves.

9. Most leaves with long lesions.

-   Guthrie, W. D., F. F. Dicke, and C. R. Neiswander. 1960. Leaf and    sheath feeding resistance to the European corn borer in eight inbred    lines of dent corn. Ohio. Agric. Exp. Sta. Res. Bull. 860.

Example 2 Directed Evolution of Axmi115v02

Directed evolution has been used to improve the potency and activityprofile of Axmi115 against ECB, Hz, FAW, BCW, and VBC. To identifyregions of Axmi115 involved in insect toxicity, a number ofAxmi115/Axmi005 sequence swap variants in the C-terminal part of Axmi115were created. Twenty-one blocks of sequence divergence between Axmi115and Axmi005 were designated (see U.S. Patent Publication No. 20100004176which is herein incorporated by reference in its entirety) and thesesequence blocks in Axmi115 were replaced with the corresponding Axmi005sequence blocks. Bioassays of hybrid proteins showed that substitutionsin blocks 2, 3, 10 and 18 are linked to increased insect toxicity.

Point mutant libraries were created that targeted positions in blocks 2,3, 10 and 18. These point mutant libraries were assayed against ECB, Hz,FAW, BCW and VBC at 1.5× coverage at the 4 replicate level. Re-assayswere carried out at the 4 replicate level, and scale-ups were done atthe 16 replicate level. The following point mutants showed improvedactivity against one or more pests:

TABLE 7 Activity of Axmi115 point mutants nucleo- amino Activity Slighttide SEQ acid SEQ improved improvement in ID NO: ID NO: against activityagainst Block2 L11C7 9 23 FAW Hz, ECB, BCW Block 2 L11H6 24 FAW Hz, ECBBlock 2 L11H7 10 25 FAW Hz, ECB, BCW Block 2 L11A9 11 26 FAW ECB, BCWBlock 2 L11F9 27 ECB BCW, FAW Block 2 L11G10 12 28 Hz, FAW Block 2 L12C313 29 Hz, FAW Block 18 L12A10 14 30 FAW ECB, VBC Block 18 L12B10 31 FAWECB

These variants contain mutations in the C-terminal part. To look forsynergistic improvements with Axmi115v02 (pAX6307), the C-terminal partof several of the above mutants was cloned into Axmi115v02 (pAX6307).Scale-up assays were carried out and variants with improved activitycompared to Axmi115v02 were identified.

TABLE 8 Activity of Axmi115v02 mutants Gene ECB FAW VBC Hz BCW Stunt %Mort Stunt % Mort Stunt % Mort Stunt % Mort Stunt % Mort axmi-115 v023.50 14.84 3.75 45.31 4.00 72.27 4.00 16.67 2.08 4.17 115B2L11H6 3.6713.02 3.67 52.08 4.00 84.38 4.00 35.16 1.50 0.00 (v02) - evo27115B18L12B10 3.33 15.63 3.67 26.56 4.00 60.94 4.00 1.56 2.25 0.00(v02) - evo28 115B2L11H7 3.33 10.94 3.67 33.33 4.00 56.77 4.00 3.91 2.000.00 (v02) 115B18L12A10 3.33 16.15 3.67 27.08 4.00 60.94 4.00 0.78 2.130.00 (v02) 115B2L11F9 3.67 6.88 3.67 54.17 4.00 86.98 4.00 34.38 1.383.13 (v02) - evo29

Variant axmi115 B2L11H6 (v02) shows improved activity against H. zea,VBC, FAW. It was designated Axmi115v02(evo27). The nucleotide sequencefor Axmi115v02(evo27) is set forth in SEQ ID NO:4 and the encoded aminoacid sequence is set forth in SEQ ID NO:18.

Variant axmi115 B18L12B10 (v02) shows improvements against ECB. It wasdesignated Axmi115v02(evo28). The nucleotide sequence forAxmi115v02(evo28) is set forth in SEQ ID NO:5 and the encoded amino acidsequence is set forth in SEQ ID NO:19.

Variant axmi115 B2L11F9 (v02) shows improvements against H. zea, VBC,FAW. It was designated Axmi115v02(evo29). The nucleotide sequence forAxmi115v02(evo29) is set forth in SEQ ID NO:6 and the encoded amino acidsequence is set forth in SEQ ID NO:20.

Additional mutations were made in the AXMI115v02 sequence in theC-terminal region. Three variants were identified with improved activityrelative to AXMI115v02 (Table 9). LC50 and EC50 values were determinedfor two of these C-terminal mutants (Table 10).

AXMI115v02(EV031) showed improved mortality against FAW, soybean looper(SBL) and VBC relative to AXMI115v02. The nucleotide sequence forAxmi115v02(evo31) is set forth in SEQ ID NO:7 and the amino acidsequence is set forth in SEQ ID NO:21.

AXMI115v02(EV032) showed improved mortality against ECB and H. zearelative to AXMI115v02. The nucleotide sequence for Axmi115v02(evo32) isset forth in SEQ ID NO:8 and the amino acid sequence is set forth in SEQID NO:22.

AXMI115v02(EV038) showed improved mortality against BCW relative toAXMI115v02. The nucleotide sequence for Axmi115v02(evo38) is set forthin SEQ ID NO:47 and the amino acid sequence is set forth in SEQ IDNO:48.

TABLE 9 Activity of Axmi115v02 C-terminal mutants Gene ECB FAW VBC HzBCW Stunt % Mort Stunt % Mort Stunt % Mort Stunt % Mort Stunt % MortAxmi115v02 3.3 11.5 4.0 16.5 4.0 80.2 4.0 13.8 2.8 1.1 Axmi115v02(evo31)3.4 28.6 4.0 20.7 4.0 81.4 4.0 14.3 2.4 0.0 Axmi115v02(evo32) 3.4 30.04.0 18.2 4.0 94.4 4.0 35.0 3.0 0.0 Axmi115v02(evo38) 0.2 0.0 4.0 15.74.0 87.1 4.0 10.5 3.6 6.6

TABLE 10 LC50 and EC50 for C-terminal mutants Gene ECB FAW VBC SBL HzBCW LC50 EC50 LC50 EC50 LC50 LC50 LC50 EC50 LC50 Axmi115v02 20 μg/ml   3μg/ml 6.3 μg/ml  1.3 μg/ml 400 ng/ml 280 ng/ml 339 μg/ml 14.3 μg/ml  7.6μg/ml Axmi115v02 18 μg/ml 4.5 μg/ml 2.4 μg/ml 240 ng/ml 120 ng/ml  80ng/ml 185 μg/ml   12 μg/ml 27.3 μg/ml (evo31) Axmi115v02 12.3 μg/ml  4.3 μg/ml   6 μg/ml 400 ng/ml 520 ng/ml 520 ng/ml 42.5 μg/ml  13.3 μg/ml16.6 μg/ml (evo32) SBL = Soybean looper

Example 3 Additional Assays for Pesticidal Activity

The nucleotide sequences of the invention can be tested for theirability to produce pesticidal proteins. The ability of a pesticidalprotein to act as a pesticide upon a pest is often assessed in a numberof ways. One way well known in the art is to perform a feeding assay. Insuch a feeding assay, one exposes the pest to a sample containing eithercompounds to be tested or control samples. Often this is performed byplacing the material to be tested, or a suitable dilution of suchmaterial, onto a material that the pest will ingest, such as anartificial diet. The material to be tested may be composed of a liquid,solid, or slurry. The material to be tested may be placed upon thesurface and then allowed to dry. Alternatively, the material to betested may be mixed with a molten artificial diet, and then dispensedinto the assay chamber. The assay chamber may be, for example, a cup, adish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA).

In some embodiments, the DNA regions encoding the toxin region of thepesticidal proteins disclosed herein are cloned into the E. coliexpression vector pMAL-C4x behind the malE gene coding for Maltosebinding protein (MBP). These in-frame fusions result in MBP-Axmi fusionproteins expression in E. coli.

For expression in E. coli, BL21*DE3 are transformed with individualplasmids. Single colonies are inoculated in LB supplemented withcarbenicillin and glucose, and grown overnight at 37° C. The followingday, fresh medium is inoculated with 1% of overnight culture and grownat 37° C. to logarithmic phase. Subsequently, cultures are induced with0.3 mM IPTG overnight at 20° C. Each cell pellet is suspended in 20 mMTris-Cl buffer, pH 7.4+200 mM NaCl+1 mM DTT+protease inhibitors andsonicated. Analysis by SDS-PAGE can be used to confirm expression of thefusion proteins.

Total cell free extracts are then run over amylose column attached tofast protein liquid chromatography (FPLC) for affinity purification ofMBP-axmi fusion proteins. Bound fusion proteins are eluted from theresin with 10 mM maltose solution. Purified fusion proteins are thencleaved with either Factor Xa or trypsin to remove the amino terminalMBP tag from the Axmi protein. Cleavage and solubility of the proteinscan be determined by SDS-PAGE

Example 4 Construction of Synthetic Sequences

In one aspect of the invention, synthetic axmi sequences are generated.These synthetic sequences have an altered DNA sequence relative to theparent axmi sequence, and encode a protein that is collinear with theparent AXMI protein to which it corresponds, but lacks the C-terminal“crystal domain” present in many delta-endotoxin proteins.

In another aspect of the invention, modified versions of synthetic genesare designed such that the resulting peptide is targeted to a plantorganelle, such as the endoplasmic reticulum or the apoplast. Peptidesequences known to result in targeting of fusion proteins to plantorganelles are known in the art. For example, the N-terminal region ofthe acid phosphatase gene from the White Lupin Lupinus albus (GenebankID GI:14276838; Miller et al. (2001) Plant Physiology 127: 594-606) isknown in the art to result in endoplasmic reticulum targeting ofheterologous proteins. If the resulting fusion protein also contains anendoplasmic retention sequence comprising the peptideN-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the “KDEL”motif (SEQ ID NO:46) at the C-terminus, the fusion protein will betargeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Example 5 Transformation of Maize Cells with the Pesticidal ProteinGenes Described Herein

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to the genes of the invention in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25° C. in the dark. Toavoid unduly damaging beamed explants, they are incubated for at least24 hours prior to transfer to recovery media. Embryos are then spreadonto recovery period media, for about 5 days, 25° C. in the dark, thentransferred to a selection media. Explants are incubated in selectionmedia for up to eight weeks, depending on the nature and characteristicsof the particular selection utilized. After the selection period, theresulting callus is transferred to embryo maturation media, until theformation of mature somatic embryos is observed. The resulting maturesomatic embryos are then placed under low light, and the process ofregeneration is initiated by methods known in the art. The resultingshoots are allowed to root on rooting media, and the resulting plantsare transferred to nursery pots and propagated as transgenic plants.

Materials

DN62A5S Media Components Per Liter Source Chu's N6 Basal 3.98 g/LPhytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6 1 mL/LPhytotechnology Labs Vitamin Solution (of 1000x Stock) (Prod. No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D 1 mL/L Sigma (Prod.No. D-7299) (of 1 mg/mL Stock)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3 g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 6 Transformation of Genes of the Invention in Plant Cells byAgrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A recombinant nucleic acid molecule comprisinga nucleotide sequence encoding a polypeptide having pesticidal activity,wherein said nucleotide sequence is selected from the group consistingof: a) the nucleotide sequence set forth in any of SEQ ID NO:2, 3, 7-14and 47; b) a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of any of SEQ ID NO:16, 17, 21-31 and 48; c) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of any of SEQ ID NO:16, 17, 21-31 and 48, wherein thepesticidal activity of the polypeptide is improved or extended relativeto the pesticidal activity of SEQ ID NO:43 or
 45. 2. The recombinantnucleic acid molecule of claim 1, wherein said nucleotide sequence is asynthetic sequence that has been designed for expression in a plant. 3.The recombinant nucleic acid molecule of claim 1, wherein saidnucleotide sequence is operably linked to a promoter capable ofdirecting expression of said nucleotide sequence in a plant cell.
 4. Avector comprising the recombinant nucleic acid molecule of claim
 1. 5.The vector of claim 4, further comprising a nucleic acid moleculeencoding a heterologous polypeptide.
 6. A host cell that contains therecombinant nucleic acid of claim
 1. 7. The host cell of claim 6 that isa bacterial host cell.
 8. The host cell of claim 6 that is a plant cell.9. A transgenic plant comprising the host cell of claim
 8. 10. Thetransgenic plant of claim 9, wherein said plant is selected from thegroup consisting of maize, sorghum, wheat, cabbage, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,tobacco, barley, and oilseed rape.
 11. A transgenic seed comprising thenucleic acid molecule of claim
 1. 12. A recombinant polypeptide havingpesticidal activity, wherein said polypeptide comprises an amino acidsequence selected from the group consisting of: a) the amino acidsequence of any of SEQ ID NO:2, 3, 7-14 and 47; c) an amino acidsequence having at least 95% sequence identity to the amino acidsequence of any of SEQ ID NO:16, 17, 21-31 and 48, wherein thepesticidal activity of the polypeptide is improved or extended relativeto the pesticidal activity of SEQ ID NO:43 or
 45. 13. The polypeptide ofclaim 12 further comprising heterologous amino acid sequences.
 14. Acomposition comprising the polypeptide of claim
 12. 15. The compositionof claim 14, wherein said composition is selected from the groupconsisting of a powder, dust, pellet, granule, spray, emulsion, colloid,and solution.
 16. The composition of claim 14, wherein said compositionis prepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof bacterial cells.
 17. The composition of claim 14, comprising fromabout 1% to about 99% by weight of said polypeptide.
 18. A method forcontrolling a lepidopteran, hemipteran, coleopteran, nematode, ordipteran pest population comprising contacting said population with apesticidally-effective amount of the polypeptide of claim
 12. 19. Amethod for killing a lepidopteran, hemipteran, coleopteran, nematode, ordipteran pest, comprising contacting said pest with, or feeding to saidpest, a pesticidally-effective amount of the polypeptide of claim 12.20. A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of claim 6 under conditions in whichthe nucleic acid molecule encoding the polypeptide is expressed.
 21. Aplant or a plant cell having stably incorporated into its genome a DNAconstruct comprising a nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide having pesticidal activity, wherein thenucleotide sequence is selected from the group consisting of: a) thenucleotide sequence set forth in any of SEQ ID NO:2, 3, 7-14 and 47; b)a nucleotide sequence that encodes a polypeptide comprising the aminoacid sequence of any of SEQ ID NO:16, 17, 21-31 and 48; and c) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of any of SEQ ID NO:16, 17, 21-31 and 48, wherein thepesticidal activity of the polypeptide is improved or extended relativeto the pesticidal activity of SEQ ID NO:43 or
 45. 22. A method forprotecting a plant from a pest, comprising expressing in a plant or cellthereof a nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide having pesticidal activity, wherein thenucleotide sequence is selected from the group consisting of: a) thenucleotide sequence set forth in any of SEQ ID NO:2, 3, 7-14 and 47; b)a nucleotide sequence that encodes a polypeptide comprising the aminoacid sequence of any of SEQ ID NO:16, 17, 21-31 and 48; and c) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of any of SEQ ID NO:16, 17, 21-31 and 48, wherein thepesticidal activity of the polypeptide is improved or extended relativeto the pesticidal activity of SEQ ID NO:43 or
 45. 23. The method ofclaim 22, wherein said plant produces a pesticidal polypeptide havingpesticidal activity against a lepidopteran, hemipteran, coleopteran,nematode, or dipteran pest.
 24. A method for increasing yield in a plantcomprising growing in a field a plant of or a seed thereof having stablyincorporated into its genome a DNA construct comprising a nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingpesticidal activity, wherein the nucleotide sequence is selected fromthe group consisting of: a) the nucleotide sequence set forth in any ofSEQ ID NO:2, 3, 7-14 and 47; b) a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of any of SEQ ID NO:16,17, 21-31 and 48; and c) a nucleotide sequence that encodes apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of any of SEQ ID NO:16, 17,21-31 and 48, wherein the pesticidal activity of the polypeptide isimproved or extended relative to the pesticidal activity of SEQ ID NO:43or 45.